Over a half million of autologous vein grafts are implanted annually in the United States. However, 30-60% of the grafts fails or develops a clinically significant stenosis within the first year, causing limb loss and death. The primary cause for early vein graft failure has been identified as neointimal hyperplasia (NIH) and compelling evidence has demonstrated that TGF-2 is a driving factor for this early failure. Unfortunately, non-selective blockade of the broad TGF-2 activities has yielded limited success in attenuating neointimal hyperplasia formation, suggesting inhibition of the specific TGF-2 activities is required. A primary mechanism that dictates TGF-2 specificity is the activation of its type I receptors Alk1 or Alk5. Although Alk1 is expressed at very low level in mature endothelium (ECs) and medial smooth muscle cells (SMCs), existing evidence suggests that Alk1 is induced in ECs and neointimal SMCs during vein graft adaptation. Recent studies for angiogenesis and other pathologies have led to an emergence of new understanding, wherein TGF-2 signals through Alk1 and Alk5 to initiate opposing effects on regulating cellular biology. We therefore hypothesize that the response of the vascular wall to TGF-2 relies on the balance between Alk1- and Alk5- signaling in both ECs and SMCs. Insult to the vein graft wall tips the balance in both cell types towards Alk5 signaling that in turn inhibits the functional recovery of ECs and upholds an inflammatory/synthetic phenotype for SMCs, driving progressive NIH. Selectively blocking Alk5 signaling to restore this balance will improve the healing response and inhibit NIH. To test this hypothesis, this project aims to: 1) Define the role of Alk1 and Alk5 signaling in SMCs in regulating the phenotype of neointimal SMCs and vein graft morphology via a validated murine vein graft model and primary neointimal SMC culture;2) Evaluate the impact of the competing Alk1 and Alk5 signaling in ECs on functional recovery of the repopulated EC monolayer, modulation of neoSMC phenotype, and the resultant vein graft morphology;and 3) Examine the therapeutic effectiveness of siRNA and pharmaceutical inhibition of Alk1 or Alk5 signaling on the development of NIH in murine and human vein grafts. The CreloxP system will be utilized to induce selective deletion of Alk1 or Alk5 in ECs or SMCs in adult mice, so that vein grafts with and without EC or SMC specific Alk1 or Alk5 can be created for the evaluation of the vein graft morphology, the repair of the EC monolayer, and the inflammatory phenotype of neointimal SMCs. To facilitate the clinical translation of the new knowledge generated with these genetic approaches, specific siRNA and novel pharmacological inhibitors will be applied to inhibit Alk1 and Alk5 signaling pathways in both murine and ex vivo human vein grafts. The therapeutic effectiveness of these approaches will then be evaluated using both morphologic (e.g. NIH volume) and biologic (e.g. phenotypic properties of the neointimal cells) endpoints. Completion of these aims will not only provide new insights into the fundamentals of TGF-2 biology, but also generate novel strategies to manipulate complex biologic processes such as vein graft wall adaptation. PUBLIC HEALTH RELEVANCE: Compelling evidence suggests that selectively blocking specific TGF-2 activities is required for effective anti TGF-2 therapy to inhibit neointimal hyperplasia, the primary pathology that causes early vein graft failure. This project seeks to understand how Alk1 and Alk5 mediated TGF-2 specificity regulates neointimal hyperplasia and test whether inhibition of Alk1 or Alk5 signaling with specific siRNA or pharmacologic inhibitors can be leveraged into the clinical application to improve the long term performance of bypass vein grafts.